A number of ce11 types, including cells derived from renal proximal tubules, exhibit the ability to take up a variety of exogtenous substances across the luminal membrane (endocytosis) and store these substances in intracellular vesicles called endosomes. Endosomes possess a proton translocating ATPase (H+ ATPase) which acidifies the interior, a process known to be critical for endosomal function. Since endosomal function is important for cellular function, endosomal acidification and regulation therefore could play an important role in modulating cellular function. While the functions of endosomal pathways have been elucidated in other tissues, endosomal pathways and functions have not been defined clearly in proximal tubule cells. It is likely that there may be different populations of endosomes with different cellular functions. Since endosomal function is dependent on endosomal acidification, it is conceivable that these differences in function arise from heterogeneity of endosomal acidification. Preliminary data presented in this proposal indicate that endosomes derived from renal proximal tubule cells display heterogeneity with respect to proton transport. Furthermore, these data also indicate that cAMP can potentially regulate endosomal acidification by inhibiting the H+ATPase directly and by interacting with chloride to stimulate proton transport. Thus, the purpose of the studies proposed in this application are: 1) To determine factors which may regulate endosomal proton transport. 2) To determine the heterogeneity of endosomes. 3) To begin to determine the function of endosomal pathways in the renal proximal tubule cells. To achieve these goals, the techniques of membrane vesicle preparation, absorbance and fluorescence spectroscopy, cell culture, polyacrylamide gel electrophoresis, autoradiography, flow cytometry, electron microscopy and radiosiotope uptake will be applied. The results of these studies should provide new information regarding the contribution of endocytosis to renal proximal tubule function including acidification in this segment.